The outermost cuticle of soybean seeds: chemical composition and function during imbibition

Seeds of different cultivars of Glycine max (L.) Merr. (soybean) have strikingly different rates of water imbibition. Seeds that readily imbibe water are termed 'soft', while those that remain non-permeable, even after several days in water, are referred to as 'hard', 'stone', or 'impermeable' seeds. What prevents soybean hard seeds from taking up water? Previous work established that the initial imbibition of soft soybean seeds correlates with the presence of small cracks in the outermost cuticle that covers the seed coat, prompting a detailed analysis of soybean seed coat cutin. In this paper, it is shown that the outermost cuticle of the seed coat has an unusual chemical composition, lacking typical mid-chain-hydroxylated fatty acids but being relatively rich in other types of hydroxylated fatty acids. The cuticle of the impermeable cultivar studied contained a disproportionately high amount of hydroxylated fatty acids relative to that of the permeable ones. Moreover, a brief treatment with hot alkali released the omega-hydroxy fatty acid component of the outermost cuticle and created holes in it that caused the seeds to become permeable. This demonstrates that the outermost cuticle of the seed is the critical structure that prevents water uptake by hard seeds.     

Cracks in the palisade cuticle of soybean seed coats correlate with their permeability to water

BACKGROUND AND AIMS: Soybean (Glycine max) is among the many legumes that are well known for 'hardseededness'. This feature can be beneficial for long-term seed survival, but is undesirable for the food processing industry. There is substantial disagreement concerning the mechanisms and related structures that control the permeability properties of soybean seed coats. In this work, the structural component that controls water entry into the seed is identified. METHODS: Six soybean cultivars were tested for their seed coat permeabilities to water. To identify the structural feature(s) that may contribute to the determination of these permeabilities, fluorescent tracer dyes, and light and electron microscopic techniques were used. KEY RESULTS: The cultivar 'Tachanagaha' has the most permeable seed coat, 'OX 951' the least permeable seed coat, and the permeabilities of the rest ('Harovinton', 'Williams', 'Clark L 67-3469', and 'Harosoy 63') are intermediate. All seeds have surface deposits, depressions, a light line, and a cuticle about 0.2 microm thick overlaying the palisade layer. In permeable cultivars the cuticle tends to break, whereas in impermeable seeds of 'OX 951' it remains intact. In the case of permeable seed coats, the majority of the cracks are from 1 to 5 micro m wide and from 20 to 200 micro m long, and occur more frequently on the dorsal side than in other regions of the seed coat, a position that correlates with the site of initial water uptake. CONCLUSIONS: The cuticle of the palisade layer is the key factor that determines the permeability property of a soybean seed coat. The cuticle of a permeable seed coat is mechanically weak and develops small cracks through which water can pass. The cuticle of an impermeable seed coat is mechanically strong and does not crack under normal circumstances.     

Anatomical and chemical characteristics of the seed coat of <i>Medicago sativa</i> L. (alfalfa) cv. Baralfa 85 seeds and their association with seed dormancy

Seeds of alfalfa (Medicago sativa L.) can exhibit seedcoat imposed dormancy, which produces hard seeds within a seed lot. These seeds do not germinate because they do not imbibe water due to a barrier to water entry in the seed coat. The aim of this work was to analyze the anatomical and chemical characteristics of the testa of alfalfa seeds with respect to water permeability levels. The anatomy of seeds of the cv. Baralfa 85 was studied and structural substances, polyphenols, tannins and cutin present in the testa of seeds of different water permeability levels were determined. The anatomical characteristics of the seed coat and the proportions of components were found to determine the permeability level of the seed coat, an aspect that is associated with the physical seed dormancy level. Anatomically, increased thickness of the testa was associated with a lower permeability level. The difference may be attributed to the variation in cuticle thickness, length of macrosclereids and thickness of the cell wall, and presence and development of osteosclereids. From the physiological and chemical points of view, the mechanism of physical dormancy of the testa is explained by a greater amount of components that repel water and cement the cell wall, such as polyphenols, lignins, condensed tannins, pectic substances, and a lower proportion of cellulose and hemicellulose.     

Deposition and localization of lipid polyester in developing seeds of Brassica napus and Arabidopsis thaliana

Mature seeds of Arabidopsis thaliana and Brassica napus contain complex mixtures of aliphatic monomers derived from non-extractable lipid polyesters. Most of the monomers are deposited in the seed coat, and their compositions suggest the presence of both cutin and suberin layers. The location of these polyesters within the seed coat, and their contributions to permeability of the seed coat and other functional properties are unknown. Polyester deposition was followed over Brassica seed development and distinct temporal patterns of monomer accumulation were observed. Octadecadiene-1,18-dioate, the major leaf cutin monomer, was transiently deposited. In contrast, the saturated dicarboxylates maintained a constant level during seed desiccation, whereas the fatty alcohols and saturated omega-hydroxy fatty acids continually increased. Dissection and analysis of Brassica seed coats showed that suberization is not specific to the chalaza. Analysis of the Arabidopsis ap2-7 mutant suggested that suberin monomers are preferentially associated with the outer integument. Several Arabidopsis knockout mutant lines for genes involved in polyester biosynthesis (att1, fatB and gpat5) were examined for seed monomer load and composition. The variance in polyester monomers of these mutants is correlated with dye penetration assays. Furthermore, stable transgenic plants expressing promoter::YFP fusions showed ATT1 promoter activity in the inner integument, whereas GPAT5 promoter is active in the outer integument. Together, the Arabidopsis data indicated that there is a suberized layer associated with the outer integument and a cutin-like polyester layer associated with the inner seed coat.     

Control of seed coat thickness and permeability in soybean : a possible adaptation to stress

Although the seed coat, through its thickness and permeability, often regulates seed germination, very little is known about the control of its development. Using soybean (Glycine max [L.] Merrill) explants, podbearing cuttings in which defined solutions can be substituted for the roots, we have demonstrated that cytokinin and mineral nutrients moving through the xylem can control soybean seed coat development. Lack of cytokinin and minerals in the culture solution, causes a thicker, less permeable seed coat to develop. The seeds with thickened coats will imbibe water rapidly if scarified; furthermore, these scratched seeds also germinate and produce normal plants. Inasmuch as stress (e.g. drought) decreases mineral assimilation and cytokinin production by the roots, the resulting delay in germination could be an adaptive response to stress.     

A Single-Nucleotide Polymorphism in an Endo-1,4-β-Glucanase Gene Controls Seed Coat Permeability in Soybean

Physical dormancy, a structural feature of the seed coat known as hard seededness, is an important characteristic for adaptation of plants against unstable and unpredictable environments. To dissect the molecular basis of qHS1, a quantitative trait locus for hard seededness in soybean (Glycine max (L) Merr.), we developed a near-isogenic line (NIL) of a permeable (soft-seeded) cultivar, Tachinagaha, containing a hard-seed allele from wild soybean (G. soja) introduced by successive backcrossings. The hard-seed allele made the seed coat of Tachinagaha more rigid by increasing the amount of β-1,4-glucans in the outer layer of palisade cells of the seed coat on the dorsal side of seeds, known to be a point of entrance of water. Fine-mapping and subsequent expression and sequencing analyses revealed that qHS1 encodes an endo-1,4-β-glucanase. A single-nucleotide polymorphism (SNP) introduced an amino acid substitution in a substrate-binding cleft of the enzyme, possibly reducing or eliminating its affinity for substrates in permeable cultivars. Introduction of the genomic region of qHS1 from the impermeable (hard-seeded) NIL into the permeable cultivar Kariyutaka resulted in accumulation of β-1,4-glucan in the outer layer of palisade cells and production of hard seeds. The SNP allele found in the NIL was further associated with the occurrence of hard seeds in soybean cultivars of various origins. The findings of this and previous studies may indicate that qHS1 is involved in the accumulation of β-1,4-glucan derivatives such as xyloglucan and/or β-(1,3)(1,4)-glucan that reinforce the impermeability of seed coats in soybean.     

Patterns and kinetics of water uptake by soybean seeds

Soybean [Glycine max (L.) Merr.] plants produce some seeds (called stone or impermeable seeds) that do not take up water for long periods of time. The present investigation confirmed that the stone seed trait is a feature of the seed coat: isolated embryos from both stone and permeable seeds took up water equally quickly. A whole, permeable seed typically imbibed water initially through its dorsal side, forming wrinkles in the seed coat and delivering water to the underlying cotyledons. Later, some lateral movement of water through the coat occurred, presumably through the air spaces of the osteosclereid layer. Imbibition by seeds was a two-phase process, the first dominated by hydration of the seed coat and the second by hydration of the cotyledons, which was rate-limited by the coat. When hydrated, coats of stone seeds were permeable to water but their hydraulic conductivity, as measured with a pressure probe, was smaller than that of coats from permeable seeds by a factor of five. Hydrated coats of both permeable and stone seeds showed weak osmometer properties.     

Development of plant cuticles: fine structure and cutin composition of Clivia miniata Reg. leaves

The fine structure and monomeric composition of the ester-cutin fraction (susceptible to BF₃/CH₃OH transesterification) of the adaxial leaf cuticle of Clivia miniata Reg. were studied in relation to leaf and cuticle development. Clivia leaves grow at their base such that cuticle and tissues increase in age from the base to the tip. The zone of maximum growth (cell expansion) was located between 1 and 4 cm from the base. During cell expansion, the projected surface area of the upper epidermal cells increased by a factor of nine. In the growth region the cuticle consists mainly of a polylamellate cuticle proper of 100–250 nm thickness. After cell expansion has ceased both the outer epidermal wall and the cuticle increase in thickness. Thickening of the cuticle is accomplished by interposition of a cuticular layer between the cuticle proper and the cell wall. The cuticular layer exhibits a reticulate fine structure and contributes most of the total mass of the cuticle at positions above 6 cm from the leaf base. The composition of ester cutin changed with the age of cuticles. In depolymerisates from young cuticles, 26 different monomers could be detected whereas in older ones their number decreased to 13. At all developmental stages, 9,16-/10,16-dihydroxyhexadecanoic acid (positional isomers not separated), 18-hydroxy-9-octadecenoic acid, 9,10,18-trihydroxyoctadecanoic acid and 9,10-epoxy-18-hydroxyoctadecanoic acid were most frequent with the epoxy alkanoic acid clearly predominating (47% at 16 cm). The results are discussed as to (i) the age dependence of cutin composition, (ii) the relationship between fine structure and composition, (iii) the composition of the cuticle proper, the cuticular layer and the non-depolymerizable cutin fraction, and (iv) the polymeric structure of cutin.Abbreviations CL cuticular layer - CP cuticle proper - MX cutin polymer matrix     

Seed coat imposed dormancy: Histochemistry of the region controlling onset of water entry into Sida spinosa seeds

Fresh Sida spinosa L. seeds do not imbibe water and germinate because the seed coat is impermeable to water. Seeds imbibe water when a portion of the coat in the chal-azal area separates from an underlying layer of subpalisade cells. Thin-walled subpalisade cells are limited to the area beneath the chalazal area. Palisade cells in the coat region that become permeable are tall, only lightly lignified, contain abundant hemicelluiose and have large cell lumena. Palisade cells in regions of the coat that remain impermeable to water are short, have heavily lignified thick walls, small cell lumena and are tightly packed. We propose that Sida spinosa seed coat dormancy is released when moisture eventually reaches the hygroscopic hemicellulose deposits in cell lumena in the lightly lignified palisade cells of the chalazal region. The subsequent expansion of the palisade cells causes the thin-walled subpalisade cells to break, resulting in separation of palisade from subpalisade cells and free passage of water through the exposed surface to the embryo, culminating in germination.     

Isolation of a gene encoding Arabidopsis membrane-associated acyl-CoA binding protein and immunolocalization of its gene product

Until recently, only cytosolic acyl-CoA binding proteins (ACBPs) have been characterized. The isolation of an Arabidopsis thaliana cDNA encoding a novel membrane-associated ACBP that accumulates in developing seeds, designated ACBP1, has provided evidence for the existence of membrane-associated forms of ACBPs (Chye, 1998, Plant Mol. Biol. 38, 827-838). We now report on the isolation of its corresponding gene from an A. thaliana Columbia genomic library using the ACBP1 cDNA as a hybridization probe. Nucleotide sequence analysis of Arabidopsis ACBP1 showed that its promoter lacks a TATA box, resembling the promoters of rat, Drosophila and human genes encoding cytosolic ACBP and suggesting that it is a housekeeping gene. We show by Western blot analysis that ACBP1 expression in developing seeds coincides with lipid deposition and that homologues of membrane-associated ACBP1 exist in other plants. Using light microscopy, we show that ACBP1 is strongly expressed in the embryo at the cotyledons, hypocotyl, procambium of the axis and in most peripheral cells of the cotyledons and hypocotyl. Immunogold labelling localized ACBP1 to vesicles, to the plasma membrane especially at epidermal cells of heart, torpedo and cotyledonary stage embryos, and to the cell wall of the outer integument cells at the seed coat. Our results suggest that ACBP1 is involved in intermembrane lipid transport from the ER via vesicles to the plasma membrane where it could maintain a membrane-associated acyl pool; its immunolocalization to the cell wall of outer integument cells at the seed coat suggests a role in cuticle and cutin formation.     

Investigations into seed dormancy in Grevillea linearifolia, G. buxifolia and G. sericea: anatomy and histochemistry of the seed coat

BACKGROUND AND AIMS: Seeds of east Australian Grevillea species generally recruit post-fire; previous work showed that the seed coat was the controller of dormancy in Grevillea linearifolia. Former studies on seed development in Grevillea have concentrated on embryology, with little information that would allow testing of hypotheses about the breaking of dormancy by fire-related cues. Our aim was to investigate structural and chemical characteristics of the seed coat that may be related to dormancy for three Grevillea species. METHODS: Seeds of Grevillea linearifolia, Grevillea buxifolia and Grevillea sericea were investigated using gross dissection, thin sectioning and histochemical staining. Water movement across the seed coat was tested for by determining the water content of embryos from imbibed and dry seeds of G. sericea. Penetration of intact seeds by Lucifer Yellow was used to test for internal barriers to diffusion of high-molecular-weight compounds. KEY RESULTS: Two integuments were present in the seed coat: an outer testa, with exo-, meso- and endotestal (palisade) layers, and an inner tegmen of unlignified sclerenchyma. A hypostase at the chalazal end was a region of structural difference in the seed coat, and differed slightly among the three species. An internal cuticle was found on each side of the sclerenchyma layer. The embryos of imbibed seeds had a water content six times that of dry seeds. Barriers to diffusion of Lucifer Yellow existed at the exotestal and the endotestal/hypostase layers. CONCLUSIONS: Several potential mechanisms of seed coat dormancy were identified. The embryo appeared to be completely surrounded by outer and inner barriers to diffusion of high-molecular-weight compounds. Phenolic compounds present in the exotesta could interfere with gas exchange. The sclerenchyma layer, together with strengthening in the endotestal and exotestal cells, could act as a mechanical constraint.     

Aliphatic composition of cutin from inner seed coat of apple

Lithium aluminum deuteride reduction released aliphatic monomers from the inner seed coat fraction but not from the outer seed coat fraction of mature apples. These monomers were identified by GC/MS and the results indicate that the inner coat of apple seed contains a cutin polymer with the major monomer acids being 18-hydroxyoctadec-9-enoic (31%), 9,10-epoxy-18-hydroxyoctadecanoic (28%) and 9,10,18-trihydroxyoctadecanoic (20 %). The monomer composition of this seed coat cuticular polymer was very similar in seeds taken from freshly harvested fruit and in those taken from fruit which had been stored at 4° for 6 months.     

A subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats

During Arabidopsis seed development large quantities of mucilage, composed of pectins, are deposited into the apoplast underneath the outer wall of the seed coat. Upon imbibition of mature seeds, the stored mucilage expands through hydration and breaks the outer cell wall that encapsulates the whole seed. Mutant seeds carrying loss-of-function alleles of AtSBT1.7 that encodes one of 56 Arabidopsis thaliana subtilisin-like serine proteases (subtilases) do not release mucilage upon hydration. Microscopic analysis of the mutant seed coat revealed no visible structural differences compared with wild-type seeds. Weakening of the outer primary wall using cation chelators triggered mucilage release from the seed coats of mutants. However, in contrast to mature wild-type seeds, the mutant's outer cell walls did not rupture at the radial walls of the seed coat epidermal cells, but instead opened at the chalazal end of the seed, and were released in one piece. In atsbt1.7, the total rhamnose and galacturonic acid contents, representing the backbone of mucilage, remained unchanged compared with wild-type seeds. Thus, extrusion and solubility, but not the initial deposition of mucilage, are affected in atsbt1.7 mutants. AtSBT1.7 is localized in the developing seed coat, indicating a role in testa development or maturation. The altered mode of rupture of the outer seed coat wall and mucilage release indicate that AtSBT1.7 triggers the accumulation, and/or activation, of cell wall modifying enzymes necessary either for the loosening of the outer primary cell wall, or to facilitate swelling of the mucilage, as indicated by elevated pectin methylesterase activity in developing atsbt1.7 mutant seeds.     

Development and structure of the seed of Ozoroa paniculosa (Anacardiaceae) and taxonomic notes

The anatropous, bitegmic and crassinucellar ovule has a nuclear endosperm development. It is further characterized by a hypostase sensu lato. This hypostase being an integral part of the chalaza undergoes a secondary extension with it. At maturity the exalbuminous seed is partially pachychalazal and therefore two anatomically distinct larger parts can be distinguished in the mature seed coat. An endotegmen typifies the integumentary seed coat, while a saddle-shaped hypostase characterizes the chalazal seed coat. This seed coat shows several characteristics of the typical anacardiaceous pachychalazal seed. The cotyledons store lipids and protein as nutrient reserveS. A well-developed cuticle, cuticular layer, cutin and callose in the hypostase cell walls, as well as tannin-like deposits in the seed coat, protect the physiologically ripe seed against dehydration.     

Immunocytolocalization of extensin in developing soybean seed coats by immunogold-silver staining and by tissue printing on nitrocellulose paper

In soybean seed coats the accumulation of the hydroxyproline-rich glycoprotein extensin is regulated in a developmental and tissue- specific manner. The time course of appearance of extensin during seed development was studied by Western blot analysis and by immunogold- silver localization. Using these techniques extensin was first detected at 16-18 d after anthesis, increasing during development to high levels at 24 d after anthesis. Immunogold-silver localization of extensin in the seed coat showed marked deposition of the glycoprotein in the walls of palisade epidermal cells and hourglass cells. The immunolocalization of extensin in developing soybean seeds was also made by a new technique--tissue printing on nitrocellulose paper. It was found that extensin is primarily localized in the seed coat, hilum, and vascular elements of the seed.     

Anatomical and biochemical changes in the composition of developing seed coats of annatto (Bixa orellana L.)

Seeds of Bixa orellana (L.) have a sclerified palisade cell layer, which constitutes a natural barrier to water uptake. In fact, newly fully developed B. orellana seeds are highly impermeable to water and thereby dormant. The purpose of this work is to investigate, from a developmental point of view, the histochemical and physical changes in the cell walls of the seed coat that are associated with the water impermeability. Seed coat samples were analyzed by histochemical and polarization microscopy techniques, as well as by fractionation/HPAEC-PAD. For histochemical analysis the tissue samples were fixed, dehydrated, embedded in paraffin and the slides were dewaxed and tested with appropriate stains for different cell wall components. Throughout the development of B. orellana seeds, there was a gradual thickening of the seed coat at the palisade region. This thickening was due to the deposition of cellulose and hemicelluloses in the palisade layer cell walls, which resulted in a highly water impermeable seed coat. The carbohydrate composition of the cell walls changed dramatically at the late developmental stages due to the intense deposition of hemicelluloses. Hemicelluloses were mainly deposited in the outer region of the palisade layer cell walls and altered the birefringent pattern of the walls. Xylans were by far the most abundant hemicellulosic component of the cell walls. Deposition of cellulose and hemicelluloses, especially xylans, could be responsible for the impermeability to water observed in fully developed B. orellana seeds.     

种子半透层研究进展

种子半透层是指在一些植物种的种子中存在的一层半透性组织,其允许种子内外水分和气体的通透,而限制或阻碍溶质的交换。种子半透层存在于种皮或胚乳部位,通常由纤维素、角质、软木脂或胼胝质等组成。半透层随着种子的发育而逐渐形成,其存在位置和化学组成具有物种多样性。半透层在种子发育、萌发和活力检测中具有重要价值。该文主要对近年来国内外有关种子半透层的特性、位置、化学组成和研究方法等方面的研究进展进行综述,并分析了目前存在的问题,提出了今后研究方向。     

Studies on Mature Seeds of Cuscuta pedicellata and C. campestris by Electron Microscopy

The seeds of Cuscuta pedicellata have been investigated by transmissionand scanning electron microscopy. Additional observations havebeen made on seeds of C. campestris by SEM only. The seed coatconsists of an outer single epidermis, two different palisadelayers, and an inner multiparenchyma layer. The outer epidermalwall in C. pedicellata has a thick cuticle and zones rich inpectic substances. The thicker ‘U-shaped’ cell wallsin the outer palisade layer are strengthened by a wall layerof hemicellulose. The inner palisade layer has thick walledcells with a ‘light line’. The inner cell wall ofthe compressed multiparenchyma layer has a thin cuticle. A fairlythick cuticle is positioned directly on the endosperm surface.The aleurone cell walls are different from the remaining endospermwalls. The latter are thick and believed to be of galactomannans.There is a ‘clear’ zone between the plasmalemmaand the cell wall in the aleurone cells. The embryo cells arepacked with lipids and proteins. In Cuscuta campestris mostendosperm has been absorbed during the seed development. Theembryo apex has two minute leaf primordia. The features of theCuscuta seeds are discussed in relation to functional and environmentalconditions. Cuscuta pedicellata, Cuscuta campestris, seed, seed coat, cuticle, cell walls, endosperm, aleurone cells, galactomannan, embryo, TEM, SEM     

Structure and Histochemistry of Embryo Envelope Tissues in the Mature Dry Seed and Early Germination of Phacelia tanacetifolia

The embryo envelope tissues in both mature dry seed and duringearly germination of Phacelia tanacetifolia were investigatedby bright-field and fluorescence light microscopy and scanningelectron microscopy. The ruminate seed had an irregularly reticulatesurface owing to the presence of polygonal areas, correspondingto the cells of the seed coat. The raised margins of these cellsjoined at the lobe tips, where radially arranged thickeningsoccurred. The unitegmic seed coat was made up of three distinctlayers: the frayed outer layer, the middle layer with portionsrising outwards to form the radial thickenings, and the innerlayer, the thickness of which was greatest in the micropylarzone. The endosperm tissue had two regions, the micropylar andthe lateral endosperm, which differed in polysaccharide composition,thickness and metachromasy intensity, and presence (in the lateralendosperm) or absence (in the micropylar endosperm) of birefringenceof the cell walls. Moreover, in the micropylar region, wherethe embryo suspensor remnant was found, Ca-oxalate crystalswere scarce or absent. The presence of a partially permeablecuticle covering the seed endosperm was observed. Incubationof seeds in Lucifer Yellow CH indicated that water was ableto penetrate quickly into the seed coat along the pathway formedby the radial thickenings, the raised margins of the polygonalcells and the middle layer. Afterwards, LY-CH readily infiltratedthe apical portions of the seed lobes and then the whole endosperm.Following imbibition, morphological changes were found in themicropylar endosperm, such as the initial digestion of proteinbodies. In addition, both in the seed coat and in the endosperm,a weaker fluorescence, probably due to leaching of polyphenolicsubstances, was observed. Once the seed coat was broken at themicropylar end of the seed, the endosperm cap surrounding theradicle tip had to be punctured by it so that complete germinationcould occur. Weakening and rupture of the micropylar endospermare briefly discussed. Copyright 2000 Annals of Botany Company Phacelia tanacetifolia, seed coat, micropylar endosperm, endosperm cap, early germination, structure, histochemistry